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WHO/BS/2018.2349 2

ENGLISH ONLY 3

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Guidelines for the safe development and production of vaccines 5

to human pandemic influenza viruses and viruses with 6

pandemic potential 7

(Proposed revision of WHO TRS No. 941, Annex 5; H1N1 specific update 2009; and H7N9 8

update 2013) 9

10

NOTE: 11

This document has been prepared for the purpose of inviting comments and suggestions on the 12

proposals contained therein, which will then be considered by the Expert Committee on Biological 13

Standardization (ECBS). Publication of this draft is to provide information about the proposed 14

Guidelines for the safe development and production of vaccines to human pandemic influenza 15

viruses and viruses with pandemic potential to a broad audience and is intended to improve 16

transparency of the consultation process. 17

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The text in its present form does not necessarily represent the final conclusions of the Expert 19

Committee. Written comments proposing modifications to this text MUST be received by 20

28 September 2018 entered in the Comment Form (available separately), and should be 21

addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention: Department 22

of Essential Medicines and Health Products (EMP). Comments may be submitted electronically to 23

the Responsible Officer: Dr Tiequn Zhou at email: [email protected]. 24

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The outcome of the deliberations of the ECBS will be published in the WHO Technical Report 26

Series. The final agreed formulation of the document will be edited to be in conformity with the 27

second edition of the WHO style guide (KMS/WHP/13.1). 28

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mailto:[email protected]

WHO/BS/2018.2349

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© World Health Organization 2018 3 All rights reserved. 4 5 This draft is intended for a restricted audience only, i.e. the individuals and organizations having received this draft. 6 The draft may not be reviewed, abstracted, quoted, reproduced, transmitted, distributed, translated or adapted, in 7 part or in whole, in any form or by any means outside these individuals and organizations (including the 8 organizations' concerned staff and member organizations) without the permission of the World Health Organization. 9 The draft should not be displayed on any website. 10 11 Please send any request for permission to: 12 13 Dr Ivana Knezevic, Technologies Standards and Norms, Department of Essential Medicines and Health Products, 14 World Health Organization, CH-1211 Geneva 27, Switzerland. Email: [email protected]. 15 16 The designations employed and the presentation of the material in this draft do not imply the expression of any 17 opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, 18 city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps 19 represent approximate border lines for which there may not yet be full agreement. 20 21 The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or 22 recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. 23 Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. 24 25 All reasonable precautions have been taken by the World Health Organization to verify the information contained in 26 this draft. However, the printed material is being distributed without warranty of any kind, either expressed or implied. 27 The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health 28 Organization be liable for damages arising from its use. 29 30 This draft does not necessarily represent the decisions or the stated policy of the World Health Organization. 31 32

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WHO/BS/2018.2349

Contents 1

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1. Introduction 3

2. Scope 4

3. Terminology 5

4. Hazard identification 6

5. Safety testing of candidate vaccine viruses 7

6. Risk assessment and management 8

9

Authors and acknowledgements 10

References 11

Appendix 1. Testing for attenuation of influenza vaccine viruses in ferrets 12

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WHO/BS/2018.2349

Abbreviations 1

BSL Biosafety level

CVV Candidate vaccine virus

EID Egg infectious dose

EMA

FFP

European Medicines Agency

Filtering face piece (mask)

GISRS (WHO's) Global Influenza Surveillance and Response System

GMP Good manufacturing practice

HA Haemagglutinin

HEPA High-efficiency particulate air (filtration)

HPAI High pathogenic avian influenza (virus)

IVPI

IVPP

(Chicken) Intravenous pathogenicity index

Influenza viruses with pandemic potential

LAIV

LPAI

Live attenuated influenza vaccine

Low pathogenic avian influenza (virus)

MDCK Madin Darby Canine Kidney (cells)

NA Neuraminidase

OIE World Organisation for Animal Health

PFU Plaque forming unit

PPE Personal protective equipment

PR8 Influenza A/Puerto Rico/8/34 virus (A/PR/8/34)

RG Reverse genetics

TCID Tissue culture infectious dose

2

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WHO/BS/2018.2349

1. Introduction 1

Careful risk assessment and strict biosafety and biosecurity precautions are needed in laboratory 2

and manufacturing environments in order to ensure safe handling of human pandemic influenza 3

viruses, candidate vaccine viruses (CVVs) and influenza viruses with pandemic potential (IVPP) 4

since the uncontrolled release of such viruses could have a significant impact on public health. In 5

2005, WHO developed biosafety risk assessment and guidelines for the production and quality 6

control of human influenza pandemic vaccines in response to the pandemic threat posed by the 7

highly pathogenic avian influenza (HPAI) A(H5N1) viruses and the need to begin development of 8

vaccines. The guidance was published in the Fifty-fifth report of the Expert Committee on 9

Biological Standardization (WHO Technical Report Series [TRS], No. 941, Annex 5) (1). Since 10

the initial publication of the guidance, experience with both IVPP and pandemic viruses for the 11

development and production of CVVs has increased globally. This experience includes developing 12

and testing CVVs derived by reverse genetics (RG) from HPAI viruses, which is reflected in this 13

update. Moreover, in response to the pandemic in 2009 caused by A(H1N1)pdm09 subtype virus 14

and the emergence of low-pathogenic avian influenza (LPAI) A(H7N9) viruses that are able to 15

infect humans, causing severe disease with a high case fatality rate, the guidance in Annex 5 of the 16

Fifty-fifth report of the Expert Committee was updated by WHO (2, 3). In addition, several WHO 17

informal consultations – including the WHO Vaccine Composition Meetings, the Global Action 18

Plan for Influenza Vaccines meetings and “Switch” meetings1 on influenza vaccine response at the 19

start of a pandemic – identified testing timelines for CVVs as one of the bottlenecks to rapid 20

vaccine responses (47). Industry, regulators and laboratories of the WHO Global Influenza 21

Surveillance and Response System (GISRS) have requested revision of the guidelines. 22

In response, WHO convened a Working Group meeting on 910 May 2017 that was attended by 23

experts, including representatives of WHO collaborating centres, WHO essential regulatory 24

laboratories, national regulatory authorities for vaccine and biosafety regulation, manufacturers, 25

and the World Organisation for Animal Health (OIE). The Working Group reviewed the 26

cumulative experience, discussed the revision of TRS 941, Annex 5, and reached a consensus on 27

the outline and key elements for the revision (8). Subsequently, a draft revision was prepared and 28

posted on the WHO website for public comments. An informal consultation meeing was held by 29

WHO on 2324 April 2018 to finalize the revision of the TRS 941, Annex 5 (9). 30

1 Meetings relating to the “switch” from production of seasonal vaccine to pandemic vaccine in an emergency.

WHO/BS/2018.2349

This document follows the risk assessment scheme used in the WHO biosafety guidance for pilot-1

lot vaccine production (10). It also includes considerations relating to the greater scale of 2

production needed to supply large quantities of vaccines rapidly where risks are likely to be 3

different from those for pilot lots. This document takes into account the considerable experience 4

gained from handling HPAI viruses and those classified as low virulence for avian species but 5

highly virulent for humans. 6

2. Scope 7

This document provides guidance to CVV-testing laboratories, vaccine manufacturers and 8

national regulatory authorities on the safe development and production of human influenza 9

vaccines in response to the threat of a pandemic. The document describes international biosafety 10

expectations for pilot-scale and large-scale vaccine production and laboratory research work. It is 11

thus relevant to development and manufacturing activities. It also specifies the measures to be 12

taken to prevent or minimize the risk to workers involved in the development and production 13

processes and the release of virus into the environment, including the risk of transmission to 14

animals. Tests required to evaluate the safety of CVVs are also described in this document. The 15

document should be read in conjunction with WHO’s Laboratory biosafety manual (11). 16

The guidance presented in this document reflects greater knowledge of A(H5N1) subtype viruses 17

(and other subtypes in general) and experience gained in the development and manufacture of 18

vaccines for A(H5N1). Moreover, a great deal has been learned from experience with the 19

A(H1N1)pdm09 viruses, and from the production of vaccines against this pandemic virus. The 20

guidance is also intended to apply to threats from any IVPP (e.g. H2, H9, H7 subtype viruses, 21

etc.), which may be virulent in humans. Manufacturers and laboratories handling HPAI viruses 22

should consult their national regulatory authorities to determine whether additional biosecurity 23

measures are required. 24

There is a significant diversity in the pathogenicity of viruses used to make CVVs that are used to 25

produce human vaccines and vaccines for other mammals. The transmission and pathogenicity of 26

influenza viruses are multifactorial traits that are not completely understood (12). The 27

haemagglutinin (HA) protein is the major virulence determinant of avian influenza viruses (13). 28

Consequently, A(H5N1) HPAI viruses that cause fatal disease in humans have been used to 29

produce reassortant viruses containing an HA that has been genetically modified to generate 30

viruses of low pathogenicity for poultry. For viruses that are inherently less pathogenic for 31

humans, wild-type virus might be used directly for inactivated vaccine production (14). Thus, both 32

WHO/BS/2018.2349

reassortants derived by classical reassortment and RG (including those using synthetic nucleic 1

acid as starting material, which may or may not be genetically modified) and wild-type viruses are 2

included within the scope of these guidelines. 3

Embryonated hens’ eggs have traditionally been used for producing most influenza vaccines, but 4

cell culture techniques have also been successfully used for seasonal and pandemic vaccine 5

production (15, 16). This guidance applies to current production technologies using eggs and cell 6

culture. 7

Both inactivated vaccines and live attenuated influenza vaccines (LAIVs) are covered by these 8

guidelines. To date, most efforts to develop CVVs for pandemic vaccines have targeted 9

inactivated vaccine. However, seasonal LAIVs derived from the A/Ann Arbor/6/60 virus have 10

been licensed in North America and western Europe, while LAIVs derived from the 11

A/Leningrad/134/17/57 virus have been licensed in China, India, Russia and Thailand (17). 12

Technologies not covered by these guidelines include new generation technology platforms that 13

do not use live influenza vaccine viruses for production (e.g. expressed recombinant proteins, 14

virus-like particles, DNA- and RNA-based vaccines and vectored vaccines), although some 15

general principles may be applicable. 16

Furthermore, the guidelines on containment measures in this document should apply to all 17

facilities and laboratories that handle live influenza viruses, including not only the CVV and 18

vaccine manufacturing facilities but also the quality control laboratories of vaccine manufacturers, 19

national control laboratories and other specialist laboratories. The transport of live virus materials 20

within and between these sites must comply with international and national specifications (18). 21

Finally, risk assessments for vaccine manufacture will vary according to whether production 22

occurs during an interpandemic period or during pandemic alert or pandemic periods (19). The 23

guidelines emphasize steps to identify and minimize risks in vaccine manufacture during the 24

interpandemic period, while indicating modifications that may be appropriate in other periods. It 25

should be noted that a pandemic preparedness approach covering both inactivated influenza 26

vaccines and LAIVs (formerly called “mock-up pandemic vaccines”) during the interpandemic 27

period has been accepted by the European Medicines Agency (EMA) (20). 28

3. Terminology 29

The definitions given below apply to the terms used in these guidelines. They may have different 30

meanings in other contexts. 31

WHO/BS/2018.2349

1

Aerosol: a dispersion of solid or liquid particles of microscopic size in a gaseous medium. 2

Airlock: areas found at entrances or exits of rooms that limit air in one space from entering 3

another space. Airlocks generally have two interlocked doors and a separate exhaust ventilation 4

system. In some cases, a multiple-chamber airlock consisting of two or more airlocks joined 5

together is used for additional control. 6

Backbone donor virus: an influenza virus that provides all or some non-glycoprotein internal 7

genes to a reassortant virus. These genes may contain determinants of attenuation and/or confer 8

high growth/high yield properties to the resulting reassortant virus. 9

Biosafety committee: an institutional/organizational committee comprising individuals versed in 10

the containment and handling of infectious materials. 11

Biosafety manual: a comprehensive document describing the physical and operational practices 12

of the laboratory facility, with particular reference to infectious materials. 13

Biosafety officer: a staff member of an institution who has expertise in microbiology and 14

infectious materials and has responsibility for ensuring that the physical and operational practices 15

of various biosafety levels are carried out in accordance with the standard procedures of the 16

institution. 17

Biosecurity: the protection and control of biological materials within laboratories and production 18

facilities in order to prevent their unauthorized access, loss, theft, misuse, diversion or intentional 19

release. 20

BSL2, BSL3 or BSL4: the combination of physical and operational requirements that protect 21

personnel, the immediate work environment and the community from exposure to infectious 22

materials during vaccine manufacture and quality control testing. Detailed principles for such 23

facilities are defined in WHO’s Laboratory biosafety manual (11) and in some national 24

regulatoryguidelines. 25

BSL2 or BSL3 enhanced: the use of additional physical and/or operational precautions, above 26

those described for BSL2 or BSL3, based on a local risk assessment in consultation with the 27

competent national authority/regulatory authority. 28

Bunded (area): an area that has either a permanent or a temporary barrier which is able to 29

contain liquid and prevent leaks and spills from spreading contamination or damaging the facility 30

(bunding). 31

Decontamination: a process by which influenza viruses are inactivated to prevent adverse health 32

and/or environmental effects. 33

WHO/BS/2018.2349

EID 50: egg infectious dose 50%, a unit for measuring the infectious activity of a biological 1

product or agent that will cause infection in 50% of inoculated chicken embryos. 2

FFP2: a filtering face-piece mask that provides high-level filtering capability against airborne 3

transmissable microorganisms and generates an effective barrier to both droplets and fine aerosols 4

to 94% efficiency. 5

FFP3: a filtering face-piece device (face-fitted mask) that provides high-level filtering capability 6

against airborne transmissable microorganisms and generates an effective barrier to both droplets 7

and fine aerosols to 99% efficiency (at 95 L/min air flow). 8

Fumigation: the process whereby a gaseous chemical is applied to sterilize or disinfect surfaces 9

in an enclosed space. 10

Good Manufacturing Practice (GMP): that part of quality assurance which ensures that 11

products are produced and controlled consistently to the quality standards appropriate to their 12

intended use and as required by the marketing authorization. 13

High efficiency particulate air (HEPA) filter: (previously high efficiency particulate absorber 14

of various efficiencies). The filter must be capable of removing at least 99.97% of all airborne 15

particles with a mean aerodynamic diameter of 0.3 microns. 16

High pathogenic avian influenza (HPAI) virus: avian influenza viruses causing systemic 17

infection and mortality in chickens which, to date, are limited to subtypes H5 and H7 containing a 18

cleavage site in HA with multiple inserted amino acids (also referred to in the literature as a 19

“multibasic” or “polybasic” cleavage site, although other insertions have also been identified). The 20

designation HPAI does not refer to the virulence of these viruses in human or other mammalian 21

hosts. 22

Inactivation: the process of rendering the influenza virus nonviable by application of heat, 23

chemicals (e.g. formalin, beta-propiolactone), UV irradiation, or other means. 24

Intravenous pathogenicity index (IVPI): an indicator used to identify an avian influnza virus as 25

HPAI or LPAI on the basis of mortality and morbidity over a 10-day period following intravenous 26

inoculation of chickens with the virus. 27

Low pathogenic avian influenza (LPAI) virus: avian influenza viruses causing infections in 28

poultry leading to no disease, mild disease or moderate disease (see also IVPI). LPAI viruses 29

typically contain an HA with a single basic amino acid preceding the site of proteolytic cleavage 30

(also referred to as a “monobasic” cleavage site). The designation LPAI does not refer to the 31

virulence of these viruses in human and other mammalian hosts. 32

WHO/BS/2018.2349

N95: a respiratory protective device designed to achieve a very close facial fit and very efficient 1

filtration of airborne particles. The “N95” designation means that the respirator blocks at least 2

95% of very small (0.3 micron) test particles when fitted correctly. 3

Primary containment: a system of containment, usually a biological safety cabinet or closed 4

container, which prevents the escape of a biological agent into the work environment. 5

Respirator hood: a respiratory protective device with an integral perimeter seal, valves and 6

specialized filtration, used to protect the wearer from toxic fumes or particulates. 7

Risk assessment: a formalized and documented process for evaluating the potential risks that may 8

be involved in a projected activity or undertaking. 9

TCID50: tissue culture infectious dose 50%. A unit of infectious activity of a biological product 10

or infectious agent that causes infection in 50% of inoculated tissue cutures. 11

Validation: the documented act of proving that any procedure, process, equipment, material, 12

activity or system leads to the expected results. 13

14

4. Hazard identification 15

Hazards associated with vaccine manufacturing and laboratory testing of CVVs of pandemic 16

viruses and IVPP depend on the type of vaccine virus (reassortant or wild-type), method of 17

production (egg-based, cell culture-based or other), whether it is an inactivated, live (attenuated) 18

virus or a recombinant virus-vectored vaccine, and whether or not there are any deliberate 19

modifications of the virus for attenuation or for enhanced immunogenicity and/or increased yield. 20

Recombinant virus-vectored vaccines (e.g. modified vaccinia Ankara virus, adenovirus, Vesicular 21

stomatitis virus) use replicating recombinant constructs based on viruses other than influenza 22

virus. The nature of their transgene, shedding and the potential for recombination are outside of 23

the scope of this document, but they are important factors that might need to be considered (21, 24

22). 25

4.1 Candidate vaccine viruses 26

CVVs for live attenuated and inactivated influenza vaccines are generally produced through 27

reassortment with well-defined backbone donor viruses (e.g. human viruses A/Puerto Rico/8/34 28

[PR8], A/Ann Arbor/6/60, or A/Leningrad/134/17/57). Wild-type viruses may also be used for 29

vaccine production. Further, new backbone donor viruses for reassortment are being developed 30

and evaluated to enhance vaccine yields and other desirable properties. It is likely that a high 31

WHO/BS/2018.2349

growth reassortant will provide the basis for future pandemic vaccine development, although it is 1

conceivable that a wild-type virus could be used. 2

3

4.1.1. Reassortants 4

The genome of influenza A virus is composed of eight individual single-stranded RNA segments 5

of negative polarity. Segments 4 and 6 encode the two surface glycoproteins, HA and 6

neuraminidase (NA), respectively. The HA is the major surface antigen of the virus, and 7

antibodies directed against HA can protect from infection by neutralizing the virus. Antibodies to 8

NA can inhibit viral infectivity at different points in the replication cycle and also have a role in 9

protection from disease (23). The remaining six RNA segments ("internal protein genes") encode 10

internal structural and nonstructural viral proteins. The segmented structure of the genome allows 11

for the exchange (reassortment) of the individual RNA segments between influenza viruses upon 12

coinfection of a single cell with two or more influenza viruses. 13

The classic or conventional method for reassortment involves preparing CVVs by co-inoculation 14

of embryonated hens’ eggs or tissue culture with a WHO recommended wild-type virus and a 15

backbone donor virus with a high growth (yield) or attenuated phenotype. Co-inoculation allows 16

for reassortment of genetic segments between the two viruses. Antiserum against the surface 17

glycoproteins of the backbone donor virus is used to select a reassortant CVV, which must contain 18

the HA gene, but normally contains both the HA and NA genes of the WHO-recommended 19

vaccine virus. Amplification in eggs (or cultured cells) results in positive selection for the optimal 20

combination of internal genes providing a high-yield reassortant virus. Several weeks are usually 21

required for the production, validation and antigenic analysis of the reassortant (7). The use of a 22

CVV in vaccine production requires approval by the national regulatory authority. 23

An alternative to the conventional approach to reassortant development is the use of RG 24

methodology to produce a reassortant vaccine virus (24). This process usually incorporates into 25

plasmids the six RNA segments encoding the internal proteins of a backbone donor virus and the 26

two segments encoding the HA and NA from the WHO-recommended vaccine virus. The 27

plasmids are subsequently transfected into cells, with or without additional helper plasmids, in 28

order to generate the CVVs to be used for vaccine manufacturing. RG technology allows for the 29

direct manipulation of the influenza gene segments and can be faster than the use of classic 30

reassortment. Moreover, if an HPAI virus is used in the RG process, the HA gene can be modified 31

to remove the specific amino acid motif at the HA cleavage site that is known to convey high 32

WHO/BS/2018.2349

pathogenicity in poultry (25). The reassortant can thus be specifically designed to serve as a CVV 1

without the capacity to cause high pathogenicity in birds. The RG system has been reported to 2

produce a CVV within 912 days (26) but further analysis of the product takes additional time. 3

The distribution and receipt of the WHO-recommended vaccine virus as a source of RNA for 4

constructing RG HA and NA plasmids adds extra time to the reassortment process. These delays 5

can be minimized if the reassorting laboratories use site‐directed mutagenesis of exisiting 6

plasmids to a related virus, or by the use of synthetic DNA (27, 28). 7

8

4.1.2 Backbone donor viruses 9

Reassortant CVVs containing donor genes (except for segments 4 and 6 [HA and NA]) from 10

backbone donor viruses (PR8, A/Ann Arbor/6/60 or А/Leningrad/134/17/57) have been widely 11

used for producing seasonal influenza vaccines and pandemic A(H1N1)pdm09 vaccines. A 12

substantial body of data indicates that reassortant viruses composed of RNA segments coding for 13

HA and NA derived from a pandemic virus or IVPP and the genes coding for the internal protein 14

genes from PR8, A/Ann Arbor/6/60 or A/Leningrad/134/17/57 will have only a low probability of 15

causing harm to human health (10, 24). 16

17

PR8 is a common backbone donor virus for generating reassortant vaccine viruses as it replicates 18

to high titre in embryonated hens’ eggs. It was originally used in the late 1960s to produce “high 19

growth reassortants” and the use of such reassortants as vaccine viruses increases vaccine yield 20

many-fold (2931). Moreover, PR8 has undergone extensive passaging in mice, ferrets and 21

embryonated hens’ eggs. This has resulted in the complete attenuation of the virus, rendering it 22

incapable of replicating in humans (32, 33). Improved backbone donor viruses are being 23

developed in order to enhance yields for CVVs used to manufacture inactivated influenza virus 24

vaccines. These new donor viruses may be derivations of PR8 viruses but they may also have 25

genes from viruses other than PR8, be synthetically generated and/or be optimized for specific HA 26

and NA subtypes (26). Demonstration of adequate attenuation of CVV using new/improved 27

backbone donor viruses will be needed before approval for use (see section 5). 28

29

Some countries have licensed live attenuated seasonal influenza vaccines using reassortants with a 30

6:2 gene constellation based on donor viruses such as the A/Ann Arbor/6/60 and 31

A/Leningrad/134/17/57. The attenuated A/Ann Arbor/6/60 virus has been used as the backbone 32

for 6:2 reassortant LAIVs. Clinical studies of some 30 different vaccine viruses over a period of 33

WHO/BS/2018.2349

more than 40 years demonstrate that A/Ann Arbor/6/60-based as well as the 1

A/Leningrad/134/17/57 reassortant vaccine viruses are attenuated for humans (34, 35). These 2

donor viruses might also be used for developing pandemic influenza vaccines and an adequate 3

level of attenuation has been shown for modified reassortant viruses of various subtypes (36). For 4

each CVV derived from a new pandemic virus or IVPP, the level of attenuation should be verified 5

by testing, as described in section 5.1. A(H5N1)-specific LAIVs made from A/Ann Arbor/6/60 6

reassortants have been licensed for pandemic preparedness purposes in several countries. 7

8

4.1.3 Gene segments from wild-type viruses (WHO-recommended vaccines viruses) 9

The gene constellation of reassortant CVVs derived by traditional co-cultivation methods must be 10

determined. Reassortants with 6:2 or 5:3 gene constellations containing the HA and NA genes of 11

the wild-type strain are most common. However, reassortants containing at the minimum the HA 12

gene could be developed with different gene constellations (e.g. one or more internal genes from 13

the WHO-recommended vaccine virus). It is also possible that a mutant (non-reassortant) wild-14

type virus could be selected that has improved growth characteristics. 15

Because of their potential association with pathogencity, the genes from the wild-type virus 16

(especially the HA and NA genes) require particular attention. 17

4.1.3.1 Haemagglutinin cleavage site 18

Most HPAI viruses of the H5 and H7 subtypes contain sequences of basic amino acids at the 19

cleavage sites separating their HA1 and HA2 domains. Elimination of the HA polybasic cleavage 20

sites is associated with reduced virulence in mammalian and avian models and a low IVPI. 21

However, some wild-type LPAI viruses (e.g. A(H7N9) viruses) have caused serious human 22

disease (37) despite causing few signs of illness in poultry. 23

For reassortants derived from HPAI H5 and H7 viruses by RG, the HA should be modified so that 24

the amino acids inserted at the HA cleavage site are reduced to a single basic amino acid; 25

additional nucleotide substitutions can be introduced in the vicinity of the cleavage site in order to 26

increase the genetic stability of the created monobasic motif during large-scale vaccine 27

manufacture. Cleavage site modifications have consistently reduced pathogenicity for avian 28

embryos and poultry (38). Reassortants used for vaccine production are expected to be of low 29

pathogenicity in poultry even if based on highly pathogenic wild-type parental viruses (39). 30

However, modifying the cleavage site does not guarantee low pathogenicity in humans and other 31

mammalian species because of the presence of other virulence factors (40). 32

WHO/BS/2018.2349

4.1.3.2 Receptor specificity 1

Preferential binding of the HA to 2,6-linked terminal sialic acid residues is associated with 2

transmissibility of influenza viruses in humans (41, 42). However, viruses that preferentially bind 3

to 2,3-linked terminal sialic acid residues (e.g. A(H7N9) and A(H5N1) viruses) do not transmit 4

well between humans but may on occasion infect humans and cause serious illness (43). While 5

receptor specificity must be considered as a factor in reducing the risk of virus transmissibility and 6

causing harm to human health, modifying it is insufficient for virus attenuation. 7

8

The hazards associated with reassortants depend in part on HA receptor specificity. If a reassortant 9

has a preference for avian cell receptors (i.e. 2,3-linked terminal sialic acid), the hazard to 10

humans is considered to be lower; however, if a reassortant has a preference for mammalian cell 11

receptors (2,6-linkages; e.g. the 1957 A[H2N2] pandemic virus) or possesses both avian and 12

mammalian receptor specificities, there is a greater risk of transmissibility and human infection. 13

For A/goose/Guangdong/1/96-lineage H5 reassortants, it is anticipated that the HA will retain a 14

preference for 2,3-linked terminal sialic acid residues, so their transmissibility between humans 15

should be reduced. However, some HPAI A(H5N1) viruses (e.g. some from Egypt) have been 16

reported to exhibit increased binding to α2,6 linkages while maintaining a preference for 2,3-17

linked terminal sialic acid residues (44, 45). It is expected that A(H5N1) reassortant viruses 18

derived by RG according to WHO guidance (46) would be attenuated for humans compared to 19

wild-type H5 viruses. Nevertheless, the human lower respiratory tract contains 2,3-linked sialic 20

acid receptors and thus exposure to high doses of A(H5N1) viruses represents a risk of infection. 21

Moreover, humans are immunologically naive to H5 and many other avian subtypes, and this too 22

is an important risk factor. 23

24

It should be noted that influenza virus pathogenicity does not depend solely on HA but is a 25

polygenic trait. The 1997 A(H5N1) virus had unusual PB2 and NS1 genes that influenced 26

pathogenicity, whereas 2004 A(H5N1) viruses possessed complex combinations of changes in 27

different gene segments that affected their pathogenicity in ferrets (47, 48). Compared to HA, the 28

NA protein of influenza viruses has a less prominent role as a virulence factor. It is known that a 29

balance of HA (receptor binding) and NA (receptor destruction and virus release) activities is 30

required for efficient viral replication (49, 50). Further, specific adaptations in NA have been 31

identified that facilitate transmission from wild aquatic birds to poultry. However, specific NA 32

determinants for the adaptation to, and virulence for, humans have so far not been found, although 33

WHO/BS/2018.2349

there is some evidence that the NA can mediate HA cleavage in A(H1N1) viruses (51, 52). It is of 1

note that resistance to the viral inhibitors oseltamivir and zanamivir is caused by specific 2

mutations in either NA or HA. 3

4.1.3.3 Secondary reassortment 4

It is conceivable that reassortment between a CVV containing HA and NA from an IVPP and a 5

seasonal human wild-type influenza virus could occur during simultaneous infection of humans 6

with both viruses. For secondary reassortants to be generated, several things need to happen, 7

namely: 8

infection of a human (e.g. production staff) with the CVV; 9

simultaneously infection of the same human with a wild-type seasonal influenza virus; 10

a reassortment between the wild-type influenza virus and the reassortant virus. 11

Such a secondary reassortant may have distinct properties from the seasonal virus and might still 12

be able to replicate in humans and spread from person to person. The likelihood of such secondary 13

reassortment is considered to be low-to-negligible. However, laboratory and production facilities 14

must have biosafety control measures in place to prevent exposure of staff to live reassortant 15

viruses. In a case of accidental exposure, it is unlikely that a CVV would replicate efficiently or 16

transmit to human contacts. In over 40 years of vaccine manufacturing, there have been no 17

reported cases of influenza as a result of secondary reassortment of CVVs. 18

4.1.4 Wild-type HPAI candidate vaccine viruses 19

The use of wild-type HPAI CVVs has been confined to cell-culture-based production, which for 20

inactivated vaccines uses closed systems under high containment. Stringent biosecurity and 21

biosafety measures are required during production, analytical testing and waste disposal in order 22

to protect staff and prevent release of infectious virus into the environment. CVVs produced by 23

RG and demonstrated to be attenuated, as described in section 5, are preferred. 24

4.1.5 Other wild-type candidate vaccine viruses 25

Vaccines may also be produced from other wild-type CVVs (e.g. swine and LPAI viruses). 26

The pathogenicity of these wild-type viruses for humans cannot be predicted; some A(H7N9) 27

viruses that are of low pathogenicity in poultry have caused severe illness in humans (53). 28

Although the transmissibility of wild-type viruses with avian receptor specificity in humans is 29

likely to be low, the transmissibility of wild-type viruses with mammalian receptor specificity 30

WHO/BS/2018.2349

(e.g. swine viruses) is largely unknown and is likely depend on a number of factors, incuding 1

population immunity. 2

3

Appropriate measures should be in place to prevent exposure of staff to the CVV derived from 4

wild-type viruses because of the risk of secondary reassortment with circulating influenza viruses, 5

as described in section 4.1.3.3. 6

4.1.6 Susceptibility of candidate vaccine viruses to NA inhibitors 7

Influenza viruses/CVVs that are sensitive to NA inhibitors or other licensed drugs should be used 8

for vaccine production whenever possible. If the relationship between genotype and phenotype is 9

well known, sequence verification may be sufficient to confirm the presence of genetic motifs 10

known to be associated with drug susceptibility. Otherwise, susceptibility should be confirmed by 11

phenotypic testing. 12

13

5. Safety testing of candidate vaccine viruses 14

CVVs can be developed by GISRS laboratories, laboratories associated with GISRS that have 15

been approved by a national regulatory authority, and laboratories of vaccine manufacturers. The 16

following tests and specifications have been developed on the basis of experience gained in 17

evaluating CVVs derived from viruses of various subtypes. The safety testing required for 18

different CVVs and their proposed containment levels are summarized in Table 1. The 19

information summarized in this table should be considered as guidance; changes in the 20

requirements may be determined on a case-by-case basis by WHO and/or national authorities. For 21

CVVs developed from newly emerging IVPP, a WHO expert group will review the data from 22

safety testing and advise WHO. WHO will then provide further guidance on appropriate 23

biocontainment requirements through its expert networks such as GISRS. 24

The requirement to conduct or complete some or all of these tests prior to the distribution of a 25

CVV may be relaxed on the basis of additional risk assessments. Such assessments should 26

consider the WHO pandemic phase, evolving virological, epidemiological and clinical data in the 27

WHO pandemic phase, as well as national and international regulatory requirements on the 28

shipment and receipt of infectious substances. 29

5.1 Tests to evaluate pathogenicity of candidate vaccine viruses 30

WHO/BS/2018.2349

The recommended tests to evaluate the pathogenicity of CVVs depend on the parental wild-type 1

viruses (i.e. WHO-recommended vaccine viruses) from which they are derived (Table 1). The 2

nature of the parental viruses and the risks of the procedures involved will also determine the 3

required biocontaiment level. The tests required to evaluate CVVs are described in the following 4

sections. Some CVVs may not require complete safety testing if they are genetically similar to a 5

CVV that has already been tested – i.e. they have HAs and NAs derived from the same, or a 6

genetically closely related, wild-type virus and are nominally on the same backbone. For these 7

CVVs, it may be sufficient to confirm sequence and genetic stability as determined by a WHO 8

expert group and/or by competent national authorities. 9

5.1.1 Attenuation in ferrets 10

Ferrets have been used extensively as an ideal indicator of influenza virus virulence for humans 11

(54). Typically, seasonal influenza viruses cause mild-to-no clinical signs in ferrets, and virus 12

replication is usually limited to the respiratory tract. PR8 viruses have also been assessed in ferrets 13

and have been found to cause few or no clinical signs, with virus replication limited to the upper 14

respiratory tract (55). However, some wild-type HPAI viruses can cause severe and sometimes 15

lethal infections in ferrets (48, 56). Thus, in the absence of human data, the ferret is generally 16

considered the best model for predicting pathogenicity/attenuation in humans. The mouse is not 17

considered an appropriate model for the safety testing of influenza CVVs. 18

CVVs should be shown to be attenuated in ferrets in accordance with Table 1, except when virus-19

specific risk assessments suggest a different approach (e.g. waiving the ferret test where Table 1 20

requires it). These tests should be conducted in well-characterized and standardized ferret models 21

(e.g. by using common reference viruses, when available, from WHO collaborating 22

centres/essential regulatory laboratories for influenza). Detailed test procedures are described in 23

Appendix 1. One or more laboratories may have ferret pathogenicity data on parental wild-type 24

viruses (i.e. WHO-recommended vaccine viruses) that could be used by all testing laboratories as 25

a further benchmark for comparison. Limiting testing requirements on the wild-type viruses will 26

minimize the time delays associated with export and import of IVPP and/or pandemic viruses. 27

Assessing the transmissibility of CVVs between ferrets is not required because of the difficulties 28

of standardizing this assay across laboratories (57, 58). 29

5.1.2 Pathogenicity in chickens 30

For CVVs derived from HPAI H5 or H7 parental viruses, determination of the chicken IVPI is 31

recommended and may also be required by national authorities. The procedure should follow that 32

WHO/BS/2018.2349

described in the OIE guidance. Any virus with an index greater than 1.2, or that causes at least 1

75% mortality in inoculated chickens, is considered an HPAI virus (59). 2

3

5.1.3 The ability of virus to plaque in the presence or absence of added trypsin 4

HPAI viruses replicate in mammalian cell culture in the absence of added trypsin, whereas LPAI 5

viruses generally do not. This test is recommended for all CVVs derived from HPAI H5 or H7 6

parental viruses. It is recommended that the test be established and characterized by the use of 7

known positive and negative control viruses (60). 8

9

5.1.4 Gene sequencing 10

Gene sequencing is important for confirming virus identity and/or verifying the presence of 11

attenuating and other phenotypic markers (e.g. markers of cold adaptation and temperature 12

sensitivity in the case of LAIV CVVs). The extent of sequencing required will depend on the 13

nature of the backbone donor viruses (e.g. LAIV or PR8). 14

15

5.1.5 Genetic stability 16

Genetic stability of CVVs is generally assesed after 610 passages in relevant substrates (i.e. 17

embryonated hens’ eggs or cultured cells). Subsequent sequence analysis can verify the retention 18

(stability) of the markers of relevant phenotypic traits, where such markers are known. These tests 19

should be conducted on all CVVs, including wild-type CVVs, prepared from pandemic viruses 20

and IVPP. It may be possible to ship viruses to manufacturers prior to fully establishing their 21

genetic stability. 22

23

WHO/BS/2018.2349

Table 1. Required safety testing of different candidate vaccine viruses (CVVs) and proposed 1

containment levels for vaccine production 2

3

4

5

a Test performed by WHO GISRS or other approved laboratory. 6

b The proposed containment levels may be changed (to higher or lower containment) based on a specific risk 7

assessment. 8 c This category refers to viruses derived by RG technology such that the additional amino acids at the HA cleavage site 9

are removed. 10 d

The requirement for performance of the chicken pathogenicity test (IVPI) is dependent on national regulatory 11 requirements which are currently under review in some countries and may change. 12 e

Genetic stability testing should be performed. However, it should not delay the distribution and use of the CVV by 13 manufacturers and can be performed subsequently.

14

Category of CVV Tests needed on CVVs

a Proposed containment for

vaccine productionb

Modified reassortant

viruses derived from H5

and H7 HPAI virusesc

Ferret (5.1.1), chicken

(5.1.2)d, plaquing (5.1.3),

sequence (5.1.4), genetic

stability (5.1.5)e

BSL2 enhanced

Reassortants and modified

viruses derived from H5

and H7 LPAI viruses

Ferret (5.1.1), sequence

(5.1.4), genetic stability

(5.1.5)e

BSL2 enhanced

Reassortants and modified

viruses derived from non-

H5 or -H7 viruses



(5.1.5)e

BSL2 enhanced

Wild-type H5, H7 HPAI

viruses

Sequence (5.1.4), genetic

stability (5.1.5)e, also see

footnotesd,f

BSL3 enhancedg

Wild-type H5, H7 LPAI

viruses



(5.1.5)e, also see footnotes

d,g

BSL2 enhancedf

Wild-type non-H5 or -H7

viruses



(5.1.5)e, also see footnote

d

BSL2 enhanced

WHO/BS/2018.2349

f Testing in ferrets and IVPI are not required because the high-pathogenic phenotype is already known and the highest 1

containment level is required to work with these viruses. 2 g

If a virus-specific hazard assessment identifies that additional control measures are appropriate, the containment level 3 may be increased – for instance, for A(H7N9) viruses additional pathogenicity testing may be required. The 4 requirements for this level may be specific to a particular facility and should be assessed on a case-by-case basis by 5 the relevant competent authority. 6

7

6. Risk assessment and management 8

6.1 Nature of the work 9

Influenza vaccine production in embryonated hens’ eggs or cell culture requires the propagation of 10

live virus. In most cases, the generation of CVVs will result in viruses that are expected to be 11

attenuated in humans (10, 24). Several steps in the manufacturing process have the potential to 12

generate aerosols containing live virus. The virus concentration in aerosols will depend on the 13

specific production step; it is highest during the harvest of infectious allantoic fluid and much 14

lower during seed virus preparation and egg inoculation, both of which use small amounts of 15

liquid containing virus or very dilute virus suspensions. Appropriate biosecurity and biosafety 16

measures (e.g. the use of laminar air flows, biological safety cabinets with HEPA filtration, 17

cleaning and decontamination of equipment, waste management and spill kits) must be in place to 18

prevent accidental exposure in the work environment and the release of virus into the general 19

environment. 20

21

6.2 Health protection 22

6.2.1 Likelihood of harm to human health 23

Wild-type influenza viruses are able to infect humans and cause serious illness; however, many of 24

the viruses used for producing vaccines are CVVs in an attenuating donor backbone (e.g. 25

A/PR/8/34, A/Ann Arbor/6/60 or А/Leningrad/134/17/57) and so the resulting CVV will have a 26

low probability of causing harm to human health. 27

28

Vaccine manufacture requires adherence to both GMP and appropriate biosafety requirements for 29

biological products, as well as related national regulations, technical standards and guidelines. 30

GMP protects the product from the operator, and protects the operator and the environment from 31

the infectious agent, thus reducing the risk of any hazard associated with production. Reassortants 32

derived from PR8 backbone donor viruses have been used routinely for producing inactivated 33

WHO/BS/2018.2349

influenza vaccines for over 40 years. This work usually requires thousands of litres of infectious 1

egg allantoic fluid, which can create substantial aerosols of reassortant virus within manufacturing 2

plants. Although manufacturing staff may be susceptible to infection with these virus aerosols, 3

there have been no anecdotal or documented cases of work-related human illness resulting from 4

occupational exposure to the reassortant viruses described above. Similarly, reassortants derived 5

from the A/Ann Arbor/6/60 and А/Leningrad/134/17/57 viruses have been used for the production 6

of LAIV for many years, yet no anecdotal or documented cases of work-related human illness 7

related to these viruses have been reported. While no study has yet been undertaken to detect 8

asymptomatic infections caused by either PR8-derived or live-attenuated viruses, the attenuation 9

status of these CVVs continues to be supported by their excellent safety record. 10

The use of pandemic CVVs that express avian influenza genes may lead to potential consequences 11

for agricultural systems. For instance, if influenza A H5 or H7 viruses or any influenza A virus 12

with an IVPI greater than 1.2 are introduced into poultry (61), OIE must be notified of the 13

presence of infection and this could lead to the implementation of biosecurity measures aimed at 14

preventing the spread of disease (59, 61). Moreover, infection of birds other than poultry 15

(including wild birds) with influenza A viruses of high pathogenicity must also be reported to 16

OIE. 17

18

6.2.2 Vaccine production in eggs 19

Influenza vaccine has been produced in embryonated hens’ eggs since the early 1940s. Much 20

experience has been gained since then; some facilities are capable of handling large numbers of 21

eggs on a daily basis with the aid of mechanized egg handling, inoculation and harvesting 22

machines. 23

Hazards may occur during production stages and/or laboratory quality control activities prior to 24

virus inactivation. During egg inoculation the virus used is dilute and relatively small in volume. 25

When the eggs are opened to harvest the allantoic fluid, the open nature of this operation may 26

result in hazardous exposure to aerosols and spills. Afterwards the allantoic fluid is handled in 27

closed vessels and so the hazards arising from live virus during subsequent processing and virus 28

inactivation (if used) are less than during virus harvest. Collection and disposal of egg waste is 29

potentially a significant environmental hazard. Safe disposal of the waste from egg-grown 30

vaccines, both within the plant and outside, is therefore critical. 31

32

WHO/BS/2018.2349

1

6.2.3 Vaccine production in cell culture 2

For pandemic influenza vaccines produced on cell cultures, the biosafety risks associated with 3

manufacturing will depend primarily on the nature of the cell culture system. Closed systems such 4

as bioreactors usually present little or no opportunity for exposure to live virus during normal 5

operation; however, additional safety measures must be taken during procedures for adding 6

samples to the bioreactor or removing them from it. Virus production in roller bottles and cell 7

culture flasks may allow for exposure to live virus through aerosols, spills and other operations. 8

Additional risks can be associated with the inactivation and disposal of the large quantities of 9

contaminated liquid and solid waste, including cellular debris, generated by this method. 10

During passage in mammalian cells, it is possible that genetic mutations may be selected in 11

pandemic and IVPP CVVs that render them more adapted to humans. These changes are most 12

likely to occur within or close to the receptor-binding domain of the HA glycoprotein. Sequence 13

analysis may detect such changes, but whether these changes affect the ability of a mutant virus to 14

cause infection in humans is not well established. Beare et al. (32) tried to de-attenuate a PR8 15

virus by multiple passage in organ cultures of human tissue but failed. Another study, with Madin 16

Darby Canine Kidney (MDCK) cells, showed that human viruses that had retained α2,6 receptor 17

(human-like) specificity were likely to mutate to an α2,3 specificity (avian-like) upon passage as 18

this provided a replicative advantage over MDCK cells (62). Overall, the hazards arising from the 19

inherent properties of a reassortant or wild-type virus are likely to be greater than the probability 20

of the virus adapting to a more human-like phenotype in cell culture. 21

6.2.4 Hazards from the vaccine 22

Inactivated pandemic influenza vaccines present no biosafety risks beyond the manufacturing 23

process, provided that the results of the inactivation steps show complete virus inactivation, 24

rendering the virus incapable of replication. 25

26

In an interpandemic or pandemic alert period, pilot-scale live attenuated pandemic influenza 27

vaccines may be developed for clinical evaluation. The biosafety risks associated with virus 28

shedding or other unintentional release of virus into the environment following vaccination should 29

be carefully assessed. Based on this risk assessment, subjects participating in clinical trials in the 30

interpandemic or pandemic alert phases should be kept in clinical isolation. If this is not done, 31

indirect hazards for humans could arise. 32

WHO/BS/2018.2349

1

While it is very unlikely that a LAIV will be harmful to humans, an indirect potential hazard may 2

exist through secondary reassortment with a human or animal influenza virus, as discussed in 3

section 4.1.3.3. 4

Evidence to date indicates that the probability of generating secondary reassortants is very low 5

(63). Moreover, containment procedures have significantly improved over the last 40 years and 6

production staff can be vaccinated to reduce the chances of acquiring an infection with a 7

circulating wild-type seasonal virus, thus minimizing the risk of secondary reassortment. In 8

addition, appropriate personal protective equipment (PPE) can also be provided. 9

10

6.3 Environmental protection 11

6.3.1 Environmental considerations 12

Influenza A viruses are enzootic or epizootic in some farm animals (poultry, pigs and horses) and 13

some populations of wild birds – particularly birds of the families Anseriformes (ducks, geese and 14

swans) and Charadriiformes (shorebirds) (64). 15

16

Several avian influenza A viruses (especially H5 and H7) can be highly pathogenic in poultry. In 17

addition, sporadic infections by influenza A viruses have been reported in other species, including 18

farmed mink, wild whales, seals, captive populations of wild cats (tigers and leopards) (65) and 19

domestic cats (66) and dogs (67). In big cats, infection has been reported following the 20

consumption of dead chickens infected with A(H5N1) viruses. 21

22

It is expected that many IVPP will have avian receptor specificity and thus birds would 23

theoretically be most susceptible. Many studies indicate that viruses with PR8 backbones are 24

attenuated in chickens. For instance, a reassortant containing an HA with a single basic amino acid 25

at the cleavage site, an NA from the 1997 Hong Kong A(H5N1) virus and the genes coding for the 26

internal protein genes of a PR8 virus, was barely able to replicate in chickens and was not lethal 27

(68). Similarly, a 6:2 PR8 reassortant that contained a 2003 Hong Kong A(H5N1) HA did not 28

replicate or cause signs of disease in chickens (55). The removal of the multiple basic amino acids 29

at the HA cleavage site in these H5/PR8 reassortants probably played a major role in reducing the 30

risk for chickens. 31

WHO/BS/2018.2349

It is likely that the temperature-sensitive phenotype of cold-adapted vaccine viruses would limit 1

replication of these viruses in avian species due to the elevated body temperature of birds. Pigs, 2

however, have both 2,3 and 2,6-linked sialic acid receptors in abundance (69) and, in the 3

absence of direct evidence to the contrary, must be considered susceptible to most influenza A 4

viruses, including LAIV and PR8 reassortants. 5

6.4 Assignment of containment level 6

Definition of containment conditions must be based on an activity-based risk assessment, taking 7

into account the scale of manipulations, the titres of live virus and whether an activity involves 8

virus amplification. Biosafety control measures must be reconciled with rules and regulations 9

governing the manufacture and testing of medicinal products under GMP (70). It should be noted 10

that biosafety control measures apply to manipulations involving live virus; such measures no 11

longer apply once a virus has been inactivated by a validated process. 12

13

The generation of reassortant CVVs from HPAI viruses typically takes place in BSL3 enhanced 14

(or BSL4) facilities, as advised by WHO (70) or competent national authorities. Special 15

consideration should be given to the hazards associated with cell culture production and quality 16

control of vaccines made from HPAI wild-type CVVs. In view of the open nature of large-scale 17

egg-based vaccine production, it is not feasible to operate in BSL3 enhanced conditions. 18

Therefore, egg-based vaccine production from HPAI wild-type viruses is not recommended. 19

20

Because of the hazards associated with egg- and cell-culture vaccine production and quality 21

control associated with classic or RG-derived reassortant CVVs that are known to be attenuated 22

(see section 5.1), the production facility should have a BSL2 enhanced containment level, as 23

specified in section 6.5.1. Under defined circumstances, CVVs for which safety testing has not 24

yet been completed may be used in production facilities that comply with containment level 25

BSL2 enhanced production facilities with additional controls, as specified in section 6.5.2, with 26

the approval of the national regulatory authority. The parts of the facility where such work (both 27

production and quality control) is carried out should meet national and OIE requirements for 28

containment, which include biosafety and biosecurity requirements and environmental controls 29

that limit the introduction into, and spread within, animal populations (59). This should be 30

agreed with the WHO expert committee and competent national authorities (71, 72). This applies 31

WHO/BS/2018.2349

to both pilot-scale and large-scale production during the interpandemic phase and pandemic alert 1

periods (71). Any subsequent relaxation of the containment level to the standard used for 2

seasonal vaccine production must be authorized by the competent national authorities on a case-3

by-case basis after evaluating the risks. 4

5

6.5 Environmental control measures 6

Containment measures to prevent the release of live virus into the environment should be 7

established on the basis of a risk assessment specific to the virus, the production system and 8

relevant biosafety guidelines – either those of national regulatory authorities or of WHO. 9

10

Local biosafety/biosecurity regulations provide guidance on the disposal of potentially infectious 11

waste. In particular, contaminated waste from production facilities may reach very high virus 12

titres. All decontamination methods must be validated. If possible, decontamination of waste 13

should take place on site. Where this is not possible, it is the responsibility of the manufacturer to 14

contain material safely during transport prior to off-site decontamination. Guidance on regulations 15

for the transport of infectious substances is available from WHO (18) and from competent national 16

authorities. In all cases the procedures must be validated to ensure that they function at the scale of 17

manufacturing. Stringent measures to control rodents, other mammals and birds must also be in 18

place. 19

20

Each manufacturer should also assess the risk of contaminating birds, horses, pigs or other 21

susceptible animals if they are likely to be in the vicinity of the manufacturing plant. Following 22

occupational exposure, staff or other personnel entering an area potentially exposed to live virus 23

should avoid visiting pig, horse or bird facilities (e.g. farms, equestrian events, bird sanctuaries) 24

for at least 14 days following exposure. If conjunctivitis or respiratory signs and symptoms 25

suggest that influenza might develop during this 14-day period, the quarantine period should last 26

for 14 days (twice the expected time for virus shedding) after the signs and symptoms have 27

resolved (73). 28

6.5.1 Specifications for BSL2 enhanced production facilities 29

In addition to the principles for BSL2 facilities that are specified in WHO’s Laboratory biosafety 30

manual (11), specifications for BSL2 enhanced facilities include those described below. 31

WHO/BS/2018.2349

6.5.1.1 Facility 1

The facility should be designed and operated to protect the recipients of the vaccine, the staff 2

producing and testing the vaccine, the environment and the population at large. Different solutions 3

may be needed according to the risks inherent in the operation(s) conducted in the area. 4

Specialized engineering solutions will be required that may include: 5

use of relative negative pressure biosafety cabinets; 6

use of high-efficiency particulate air (HEPA) filtration of air prior to its exhaust into 7

public areas or the environment; 8

room pressure cascades designed to contain live virus safely while also protecting the 9

product (A net negative pressure between the atmosphere and areas where live virus is 10

handled can be separated by an area of positive pressure (barrier) higher than the 11

pressure in the atmosphere and areas where live virus is handled. Alternatively, a 12

negative pressure barrier can be built where live virus is trapped before it escapes into 13

the atmosphere and is removed from the trap with the air flow through HEPA filters. 14

In addition, the following decontamination procedures should take place: 15

decontamination of all waste from BSL2 enhanced (pandemic influenza vaccine) areas; 16

decontamination of all potentially contaminated areas at the end of a production 17

campaign through cleaning and validated decontamination (e.g. fumigation). 18

6.5.1.2 Personal protection 19

A range of personal protection measures should be in place, including the following: 20

Full-body protective laboratory clothing (e.g. Tyvek® disposable overalls) should be available. 21

If activities cannot be contained by primary containment and open activities are being 22

conducted, the use of respiratory protective equipment which can be checked for close facial 23

fit, such as FFP2 (e.g. N95), FFP3 (75) respirators, is required. Minimal specifications for the 24

filtering/absorbing capacity of such equipment should be met and masks, if used, must be 25

fitted properly and the correctness of fit tested. 26

All personnel, including support staff and others who may enter the production or quality 27

control areas where CVVs, pandemic viruses and IVPP are handled, should sign a written 28

document in which they agree not to have any contact with susceptible animals (e.g. ferrets or 29

farm animals, especially birds, horses and pigs) for 14 days after leaving the facility where 30

vaccine has been produced. If conjunctivitis or respiratory signs and symptoms suggesting 31

WHO/BS/2018.2349

influenza develop during this 14-day period, the period should be extended to 14 days after the 1

signs and symptoms have resolved (59). Currently the risks involved in contact with household 2

dogs and cats are not considered to be significant, but the scientific evidence on this risk is 3

sparse. 4

It is strongly recommended that staff should be vaccinated with seasonal influenza vaccines. 5

If vaccines targeting the virus in production are available and marketing authorization has been 6

received, vaccination with these vaccines is recommended for staff before large-scale vaccine 7

production commences. 8

A medical surveillance programme of staff should be established prior to manufacturing 9

activities. Antiviral medicines should always be available in case of accidental exposure. 10

Where these medicines are available only on prescription, access to prescribing 11

doctors/hospitals and to stocks of medication should be ensured. 12

6.5.1.3 Monitoring of decontamination 13

Cleaning and decontamination methods must be validated and reviewed periodically as part of a 14

master plan to demonstrate that the protocols, reagents and equipment used are effective in 15

inactivating pandemic influenza virus on all surfaces, garments of personnel, waste materials and 16

storage containers. Once decontamination protocols for influenza virus have been fully described 17

and validated, there is no need to repeat a validation study for each new influenza virus. 18

Validation studies using influenza viruses may be supplemented by studies with biological (e.g. 19

bacterial) markers selected to be more difficult to inactivate than influenza virus. 20

21

6.5.2 Specifications for BSL2 enhanced production facilities with additional controls 22

It is assumed that ferret pathogenicity testing will be conducted on all CVVs of unknown 23

pathogenicity, even given the assumptions above (section 5.1) regarding the low probability that a 24

PR8 reassortant virus or LAIV is pathogenic for humans. This assumption is based on current 25

experience related to reassortants of HA subtypes other than H1 and H3 (i.e. H5, H7 and H9). A 26

facility must meet requirements for protecting personnel who handle potentially dangerous micro-27

organisms. WHO’s Laboratory biosafety manual (11) includes a risk survey that can be 28

undertaken prior to rating a laboratory space as either BSL1, BSL2 or BSL3. Similar requirements 29

can be found in the European Directive 2000/54/EC (2007) (72) on the protection of workers from 30

WHO/BS/2018.2349

risks related to exposure to biological agents at work, and the United States Biosafety in 1

microbiological and biomedical laboratories guide (fifth edition 5) (74). 2

Large-scale vaccine manufacturing using a CVV before its safety testing is complete can be 3

considered if justified by evolving virological, epidemiologcal and clinical data as well as national 4

and international regulatory requirements regarding the shipment, receipt and handling of 5

infectious substances. 6

7

Following review of the requirements for Biosafety at BSL3, a facility that meets the criteria 8

detailed below and that has the noted operator protection in place could be considered suitable to 9

manufacture vaccine at large scale using a CVV prepared by RG or classical reassortant 10

methodology, before safety testing is complete and with the approval of the national regulatory 11

authority. 12

6.5.2.1 Facility 13

The facility should be designed and operated to meet the demands of protecting the recipient of 14

the vaccine, the staff producing and testing the vaccine, and the environment. This will require 15

specialized engineering solutions that may include the following: 16

Appropriate signage and labelling must be in place regarding the activities being carried out 17

when a virus is in use while the safety testing is being completed. 18

The facility must be designed and constructed as a contained GMP space. The surfaces and 19

finishes must comply with GMP requirements (70) that ensure they can be sealed and easily 20

cleaned and decontaminated. 21

The air cascades within the facility should be such that any live virus can be contained within 22

the work zones in which it is being used. All work with infectious virus must be conducted 23

within these contained zones. 24

Access to the contained areas must be via double-door entry airlocks. The airlocks should 25

operate at a pressure that is either lower or higher than that on either side. In this way the 26

airlocks become either a “sink” or a pressure barrier, containing the flow of air within the 27

facility. In the case where the airlocks provide a low-pressure sink, the entry and exit doors 28

should be interlocked or fitted with a suitable delay or alarm system to prevent both being 29

opened at the same time. It is also acceptable if the airlocks are part of a series of increasing 30

negative pressure. The air pressure cascade within the negative-pressure contained zone should 31

comply with GMP (i.e. higher pressures in cleanest zones) requirements for clean rooms. 32

WHO/BS/2018.2349

All supply and exhaust air must be passed through HEPA filtration while maintaining all 1

required containment and GMP conditions. Air-handling systems within the facility must be 2

rigorously assessed to ensure that they protect against potential failure. Fail-safe systems must 3

be installed wherever necessary. The facility should be constantly monitored to ensure that 4

appropriate room pressure differentials are maintained. 5

All reusable equipment should be cleaned in place, decontaminated by means of autoclaving, 6

or otherwise cleaned and decontaminated by validated, dedicated systems prior to reuse. 7

Areas of potential liquid spill, including waste treatment plants and processes, should be 8

assessed and bunded to ensure that any spill is contained. Procedures must be in place to 9

ensure that spills are contained, areas are cleaned and contaminated materials are properly 10

disposed of in order not to compromise the integrity of the facility. 11

The entry of materials into contained zones should be via separately HEPA-filtered, 12

interlocked, double-ended “pass-through cabinets” or double-ended autoclaves. 13

All facility waste, including egg waste, should be discarded via validated on-site waste 14

effluent systems or by autoclaving. Any items which pass from the external environment to the 15

manufacturing process and are later returned to the external environment (e.g. egg trays) must 16

receive special attention. Dedicated washing and decontamination of equipment and/or 17

procedures must be provided and fully validated. 18

6.5.2.2 Personal protection 19

All clothing worn outside the facility should be replaced by manufacturing-facility garments 20

on entry into the facility. 21

Gowning in areas in which live virus is handled should always include full suit, overshoes, eye 22

protection and double gloves. 23

Suitable PPE (full hood powered air-purifying respirators based on the risk assessment) should 24

be provided for all personnel working in containment areas within the manufacturing facility. 25

The hoods are to be worn at all times when the facility is in operation under these enhanced 26

biosafety requirements. 27

All facility clothing is to be removed on exit, with soiled clothing removed from the facility 28

via a decontamination autoclave or similar method. The surfaces of respirator hoods should be 29

decontaminated. 30

WHO/BS/2018.2349

Specific procedures should be developed and implemented for operation of the facility under 1

enhanced biosecurity conditions. 2

It is strongly recommended that staff are vaccinated with a seasonal influenza vaccine. In the 3

case of pandemic viruses and IVPP and before large-scale vaccine production is attempted, 4

pilot lots of vaccine may already have been produced. If they are available and if marketing 5

authorization has been received, vaccination of staff against the virus being produced is 6

recommended before large-scale production begins. 7

Procedures should be in place to provide antiviral treatment whenever warranted (e.g. 8

accidental exposure). 9

On-site occupational health and safety and medical support should be maximized by providing 10

medical consultation and training in recognizing influenza-like symptoms plus out-of-hours 11

referral to medical facilities with quarantine capabilities. 12

It is recommended staff should take showers on exiting the facility. Showers are mandatory for 13

staff who may have been accidently exposed to vaccine virus. 14

All personnel who enter production or quality control areas where live pandemic viruses and 15

IVPP may be handled should sign their agreement to a written instruction not to have contact 16

with susceptible animals (including poultry, pigs, horses, ferrets and non-human primates) for 17

14 days following departure from the facility where vaccine has been produced. If 18

conjunctivitis or respiratory signs and symptoms indicating the possibility of influenza develop 19

during this 14-day period, this period should be extended to 14 days after the signs and 20

symptoms resolve. The risks involved in contact with domestic dogs and cats are not 21

significant, but scientific evidence on this is sparse. 22

6.6 Biosafety management and implementation within a vaccine production facility 23

6.6.1 Management structure 24

Implementation of these guidelines requires that the institution employs a biosafety officer who is 25

knowledgeable about large-scale virus production and containment but whose reporting 26

responsibilities are independent of the production unit. The biosafety officer is responsible for 27

overseeing the implementation of biosafety practices, policies and emergency procedures within 28

the company or organization and should report directly to the highest management level. A 29

biosafety officer is needed in addition to a qualified person who, in some countries, has overall 30

responsibility for a medicinal product. 31

WHO/BS/2018.2349

1

A Biosafety Committee that includes representatives of the virus production and quality control 2

units should be responsible for reviewing the biosafety status of the company and for coordinating 3

preventive and corrective measures. The institutional biosafety officer must be a member of the 4

committee. The committee chairperson should be independent of both the production and quality 5

control units. The Biosafety Committee should report to the executive management of the 6

manufacturing company to ensure that adequate priority is given and resources are available to 7

implement the required measures. 8

9

6.6.2 Medical surveillance 10

Manufacturers of vaccines to protect against human pandemic influenza viruses and IVPP should 11

provide training to their occupational health professionals in recognizing the clinical signs and 12

symptoms of influenza. Company physicians, nurses and vaccine manufacturing supervisors and 13

staff must make decisions on the health of personnel who are associated with manufacturing and 14

testing of these vaccines. Local medical practitioners caring for personnel from the manufacturing 15

site should receive special training in the diagnosis and management of pandemic influenza 16

infection and should have access to rapid influenza diagnostic kits and a laboratory that performs 17

molecular diagnosis (e.g. real-time PCR) of influenza. Any manufacturer starting large-scale 18

production should have documented procedures – including diagnostic procedures and prescribed 19

treatment protocols – for dealing with influenza-like illness affecting the staff and their family 20

members. Manufacturers should ensure that staff understand their obligation to seek medical 21

attention for any influenza-like illness and to report it to the occupational health department or 22

equivalent. Manufacturers should ensure that antiviral treatment is available if warranted (e.g. in 23

the case of accidental exposure) and should have defined arrangements for advising staff with any 24

influenza-like illness. 25

26

6.6.3 Implementation 27

A comprehensive risk analysis should be conducted to define possible sources of contamination of 28

personnel or the environment that may arise from the production or testing of live influenza virus. 29

The analysis should take into account the concentration, volume and stability of the virus at the 30

site, the potential for inhalation or injection that could result from accidents, and the potential 31

consequences of a major or minor system failure. The procedural and technical measures 32

WHO/BS/2018.2349

necessary to reduce the risk to workers and to the environment should be considered as part of this 1

analysis, and the results should be documented. 2

3

A comprehensive biosafety manual or equivalent document must be published and implemented. 4

The manual should fully describe the biosafety aspects of the production process and the quality 5

control activities. It should define such items as emergency procedures, waste disposal, and the 6

safety practices and procedures that have been identified in the risk analysis. The manual must be 7

made available to all staff of the production and quality control units, and at least one copy must 8

be present in the containment area(s). The manual should be reviewed and updated when changes 9

occur and in and in any case at least every two years. 10

11

Comprehensive guidelines outlining the response to biosafety emergencies, spills and accidents 12

should be prepared and should be made available to key personnel both for information and for 13

coordination with emergency response units. Rehearsals of emergency response procedures are 14

helpful. The guidelines should be reviewed and updated at a defined frequency (e.g. annually). 15

16

The implementation of the appropriate biosafety level status in the production and testing facilities 17

should be verified through an independent assessment. National requirements concerning 18

verification mechanisms should be in place and must be followed. 19

20

Authors and acknowledgements 21

The first draft was prepared by Dr G. Grohmann, University of Sydney, Australia and Dr T. Zhou, 22

World Health Organization, Switzerland with critical input from Dr O.G. Engelhardt, National 23

Institutes for Biological Standards and Control, Medicines and Healthcare Products Regulatory 24

Agency, United Kingdom; Dr J.M. Katz, Centers for Disease Control and Prevention, USA; Dr R. 25

Webby, University of Tennessee, USA and Dr J. Weir, Food and Drug Administration, USA 26

based on input from the participants of the Working Group Meeting on Revision of WHO TRS 27

941, Annex 5: WHO Biosafety Risk Assessment and Guidelines for the Production and Quality 28

Control of Human Influenza Pandemic Vaccines, held on 910 May 2017: 29

Othmar G Engelhardt, National Institute for Biological Standards and Control, Medicines and 30

Healthcare products Regulatory Agency, Potters Bar, United Kingdom; Glen Gifford; World 31

Organisation for Animal Health (OIE), France; Shigeyuki Itamura, Centre for Influenza Virus 32

WHO/BS/2018.2349

Research, National Institute of Infectious Diseases, Tokyo, Japan; Jacqueline M. Katz, WHO 1

Collaborating Centre for the Surveillance, Epidemiology and Control of Influenza, Centers for 2

Disease Control and Prevention, Atlanta, United States of America; Changgui Li, National 3

Institutes for Food and Drug Control, Beijing, People's Republic of China; John McCauley, WHO 4

Collaborating Centre for Reference and Research on Influenza, Crick Worldwide Influenza 5

Centre, The Francis Crick Institute, London, United Kingdom; Jennifer Mihowich, Centre for 6

Biosecurity, Health Security Infrastructure Branch, Public Health Agency of Canada, Ottawa, 7

Canada; Ralf Wagner, Paul-Ehrlich-Institut, Langen, Germany; Richard Webby, WHO 8

Collaborating Centre for Studies on the Ecology of Influenza in Animals, St. Jude Children's 9

Research Hospital, University of Tennessee, Memphis, United States of America; Jerry Weir, 10

Centre for Biologics Evaluation and Research, Food and Drug Administration, Maryland, United 11

States of America; Gert Zimmer, Institute of Virology and Immunology, Mittelhäusern, 12

Switzerland. Representatives of international federation of pharmaceutical manufacturers & 13

associations (IFPMA): Matthew Downham, AstraZeneca/Medimmune, United Kingdom; Lionel 14

Gerentes, Sanofi Pasteur, France; Elisabeth Neumeier, GSK Vaccines, Germany; Beverly Taylor, 15

Seqirus, United Kingdom. Representatives of developing countries vaccine manufacturers 16

network (DCVMN): Pradip Patel, Cadila Healthcare Limited, Ahmedabad, India; Kittisak 17

Poopipatpol and Ponthip Wirachwong, Government Pharmaceutical Organization, Bangkok, 18

Thailand. WHO headquarters: Mustapha Chafai, Prequalification Team, Essential Medicines and 19

Health Products (EMP) Department, Health Systems and Innovation (HIS) Cluster, World Health 20

Organization (WHO), Geneva, Switzerland; Gary Grohmann, HIS/WHO, Switzerland; Ivana 21

Knezevic, Technologies Standards and Norms (TSN) Team, EMP/HIS/WHO, Geneva, 22

Switzerland; Wenqing Zhang, High Threat Pathogens (PAT), Infectious Hazard Management 23

(IHM), Health Emergencies Programme (WHE), WHO, Geneva, Switzerland; TieQun Zhou, 24

TSN/EMP/HIS/WHO, Geneva, Switzerland. 25

Acknowledgments are due to the following experts/institutes who provided written comments in 26

response to the posting of the first draft document on the WHO Biologicals website during 27

November 2017- Feburary 2018 for the first round of public consultation: 28

Dr J. Southern, MCC South Africa; Mr D. Laframboise, Public Health Agency of Canada, 29

Canada; PT Bio Farma (Persero), Indonesia; Dr T.M. Tumpey, Centers for Disease Control and 30

Prevention, USA; Dr R. Shivji, European Medicines Agency, United Kingdom; International 31

Federation of Pharmaceutical Manufacturers & Associations comments provided by Ms P. 32

Barbosa; Dr C. Thompson and Dr M. Zambon, Public Health England, United Kingdom; 33

WHO/BS/2018.2349

Comments collected by Dr R. Donis, Biomedical Advanced Research and Development 1

Authority, USA, including comments from Mr M. Sharkey, Aveshka, Inc., Rober Fisher, USA; Dr 2

L.R. Yeolekar, Serum Institute of India; comments provided by WHO Vaccines Assessment 3

Group in Prequalifiation Team, Department of Essential Medicines and Health Products; and Dr 4

K. Kojima, Biosafety Group, World Health Organization, Switzerland. 5

A second draft was prepared by Dr G. Grohmann, University of Sydney, Australia and Dr T. 6

Zhou, World Health Organization, Switzerland with critical input from Dr O.G. Engelhardt, 7

National Institutes for Biological Standards and Control, United Kingdom; Dr J.M. Katz, Centers 8

for Disease Control and Prevention, USA; Dr R. Webby, University of Tennessee, USA and Dr J. 9

Weir, Food and Drug Administration, USA taking into consideration the outcomes of the WHO 10

meeting “Informal consultation on WHO biosafety risk assessment and guidelines for the 11

production and quality control of novel human influenza candidate vaccine viruses and pandemic 12

vaccines”, Geneva, Switzerland 2324 April 2018. Acknowledgements are due to input from the 13

following participants: Mr P. Akarapanon, Ministry of Public Health, Thailand; Dr D. Bucher, 14

New York Medical College, USA; Dr O.G. Engelhardt, National Institute for Biological Standards 15

and Control, Medicines and Healthcare Products Regulatory Agency, United Kingdom; Dr G. 16

Grohmann, University of Sydney, Australia; Dr S. Gupta, National Centre for Disease Control, 17

India; Dr S. Itamura, National Institute of Infectious Diseases, Japan; Mr D. Laframboise, Public 18

Health Agency of Canada, Canada; Dr J. McCauley, The Francis Crick Institute, United Kingdom; 19

Dr G. Pavade, World Organisation for Animal Health, France; Dr M. Sergeeva, Ministry of 20

Health of Russia, Russian Federation; Dr N.T.T. Thuy, Drug Registration Division, Viet Nam; Dr 21

R. Wagner, Paul-Ehrlich-Institut, Germany; Dr D. Wang, Chinese Center for Disease Control and 22

Prevention, China; Dr R. Webby, University of Tennessee, USA; Dr J. Weir, Food and Drug 23

Administration, USA; Dr J. Yoon, Ministry of Food and Drug Safety, Republic of Korea; Dr M. 24

Zambon, Public Health England, United Kingdom. Representatives of the International 25

Federation of Pharmaceutical Manufacturers & Associations : Dr M. Downham, 26

AstraZeneca/Medimmune, United Kingdom; Dr L. Gerentes, Sanofi Pasteur, France; Dr E. 27

Neumeier, GSK Vaccines, Germany; Dr B. Taylor, Seqirus, United Kingdom. Representatives of 28

Developing Countries Vaccine Manufacturers Network (including WHO pandemic influenza 29

Global Action Plan manufacturers): Dr L. Yeolekar, Serum Institute of India, India; Dr I.G. 30

Setiadi, Biofarma, Indonesia; Ms V. Fongaro Botosso, Instituto Butantan, Brazil; Dr D. Huu Thai, 31

Institute of Vaccines and Medical Biologicals, Viet Nam; Dr K. Kojima, World Health 32

Organization, Switzerland; Dr I. Knezevic, World Health Organization, Switzerland; Dr J. Shin, 33

WHO/BS/2018.2349

World Health Organization Regional Office for the Western Pacific, Philippines; Dr W. Zhang, 1

World Health Organization, Switzerland; and Dr T. Zhou, World Health Organization, 2

Switzerland. 3

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WHO/BS/2018.2349

Appendix 1 1

Testing for attenuation of influenza CVVs in ferrets 2

3

Laboratories testing for attenuation of CVVs in ferrets should make use of a panel of 4

standard/reference viruses (“pathogenicity standards” in the following section) with defined 5

experimental outcomes for pathogenicity testing. The pathogenicity standards (to be established 6

by WHO laboratories) serve as benchmarks for the pathogenicity test in ferrets and delineate the 7

expected outcomes. The use of these standards will ensure that attenuation of CVVs is being 8

measured against common parameters independently of subtype. The CVV to be tested must show 9

parameters of pathogenicity that are below the predefined values of a high pathogenicity standard 10

and are in line with the values of an attenuation standard in order to be designated as attenuated. 11

Comparative attenuation with the parental wild-type virus is not necessary in this case. However, 12

laboratories that have the capacity to evaluate attenuation of a CVV compared with the parental 13

wild-type virus can continue to do so. To account for the expected experimental variability of 14

results across different laboratories, the pathogenicity standards can be tested in ferrets at each 15

testing laboratory according to the experimental protocol shown below when establishing the 16

ferret model for pathogenicity testing and at regular intervals thereafter. The outcomes of these 17

tests should fall within the limits described for the pathogenicity standards. In cases of 18

discrepancy, a review of the ferret model should be conducted and advice should be sought from 19

experienced WHO laboratories. 20

Test virus 21

The 50% egg or tissue culture infectious dose (e.g. EID50, TCID50) or plaque-forming units (PFU) 22

of the reassortant CVV or pathogencitiy standard will be determined. The infectivity titres of 23

viruses should be sufficiently high to allow infection with 107 to 10

6 EID50, TCID50 or PFU of 24

virus and diluted not less than 1:10. Where possible, the pathogenic properties of the donor PR8 25

should be characterized thoroughly in each laboratory. 26

27

Laboratory facility 28

Animal studies with the CVV and the pathogenicity standards should be conducted in animal 29

containment facilities in accordance with the proposed containment levels shown in Table 1. For 30

untested CVVs, the biocontainment level to be used for the ferret safety test is the one shown for 31

the respective wild-type virus. In specific cases, such as for CVVs derived from synthetic DNA 32

WHO/BS/2018.2349

representing H5 and H7 HPAI viruses, the containment level may be lowered based on a virus-1

specific risk assessment. An appropriate occupational health policy should be in place. 2

3

Experimental procedure 4

Outbred ferrets 412 months of age that are serologically negative for currently circulating 5

influenza A and B viruses and the test virus strain are anaesthetized by either intramuscular 6

administration of a mixture of sedatives (e.g. ketamine [25 mg/kg] and xylazine [2 mg/kg] and 7

atropine [0.05 mg/kg]) or by suitable inhalant anaesthetics. A standard virus dose of 107 to10

6 8

EID50 (or TCID50 or PFU) in 0.51 mL of phosphate-buffered saline is used to inoculate 9

animals. The dose should be the same as that used for pathogenicity studies with the wild-type 10

parental virus, if used, or the pathogenicity standards previously characterized and regularly 11

assessed in the laboratory. The virus is slowly administered into the nares of the sedated animals, 12

reducing the risk of virus being swallowed or expelled. A group of 46 ferrets should be 13

inoculated. One group of 23 animals should be euthanized on day 3 or day 4 after inoculation 14

and samples should be collected for estimation of virus replication from the following organs: 15

spleen, intestine, lungs (samples from each lobe and pooled), brain (anterior and posterior sections 16

sampled and pooled), olfactory bulb of the brain, and nasal turbinates. If gross pathology 17

demonstrates lung lesions similar to those observed in wild-type viruses or established standards, 18

it is recommended that additional lung samples be collected and processed with haematoxylin and 19

eosin staining for histopathological evaluation. The remaining brain tissue should be collected for 20

histopathological evaluation in the event that infectious virus is detected in this tissue. The 21

remaining animals are observed for clinical signs, which may include weight loss, lethargy (based 22

on a previously published index) (1), respiratory and neurological signs and increased body 23

temperature. Collection of nasal washes from animals anaesthetized as indicated above should be 24

performed to determine the level of virus replication in the upper airways on alternate days after 25

inoculation for up to seven days. At the termination of the experiment on day 14 after inoculation, 26

a necropsy should be performed on at least two animals and organs should be collected. If signs of 27

substantial gross pathology are observed (e.g. lung lesions), the organ samples should be 28

processed as described above for histopathology. 29

30

Expected outcome 31

Clinical signs of disease, such as lethargy and/or weight loss, should be within the predefined 32

ranges of acceptable pathogenicity defined by the pathogenicity standards, and histopathology of 33

WHO/BS/2018.2349

the lungs should demonstrate attenuation when compared to wild-type viruses or established 1

standards. Viral titres of the vaccine strain in respiratory samples should be within the ranges of 2

acceptable virus replication defined by the pathogenicity standards. Replication of the CVV 3

should be restricted to the respiratory tract. Virus isolation from the brain is not expected. 4

However, detection of virus in the brain has been reported for some seasonal A(H3N2) viruses (2) 5

where virus was detected in the olfactory bulb. Consequently, if virus is detected in the anterior or 6

posterior regions of the brain (excluding the olfactory bulb) the significance of such a finding may 7

be confirmed by performing immunohistochemistry to detect viral antigen and/or 8

histopathological analysis of brain tissue collected on day 3 or day 4 and on day 14 after 9

inoculation. The detection of viral antigen and/or neurological lesions in brain tissue would 10

confirm virus replication in the brain. The presence of neurological signs and confirmatory viral 11

antigen and/or histopathology in brain tissue would indicate a lack of suitable attenuation of the 12

CVV. 13

14

A model table for reporting results of ferret tests is provided below (Table 1) with the intention of 15

harmonizing the data reporting between CVV testing laboratories. 16

Table 1. Summary of results in ferrets infected intranasally with CVV 1 Virus

Dose (EID50)

a

Number of animals

Number of animals with clinical signs to day 14 post-inoculation

Mean maximum % weight loss

Respiratory tract viral titers (log10EID50/mL or TCID50)

b

Lung lesions (day 3/4)

c,d

Lung lesions (day 14)

c,d

Detection of virus in other organ

e

Lethargy

Respiratory

Weight loss

Other (e.g. fever)

Mean peak nasal wash or nasal turbinate

Lung

CVV

Reference virus(es)

2 a Indicate whether dose is expressed as EID50, TCID50 or PFU.

3 b Indicate whether respiratory viral titres are expressed as EID50, TCID50 or PFU per mL or g. Give lower limit of detection. 4

c Score gross pathological lung lesions as -, + (≤20%), ++ (>20 and < 70%), +++ (>70%). 5

d Indicate outcome of any histopathology evaluation. 6

e Indicate organ or not detected. 7 8

References for Appendix 1 9

10

1. Reuman PD, Keely S, Schiff GM. Assessment of signs of influenza illness in the ferret model. Lab Anim Sci. 1989;42:222–32. 11

2. Zitzow LA, Rowe T, Morken T, Shieh WJ, Zaki S, Katz JM. Pathogenesis of avian influenza A (H5N1) viruses in ferrets. J Virol. 12

2002;76:4420–9. 13

14

15

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